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Many children with sickle cell disease living in sub-Saharan Africa die before reaching age 5 years. We estimate the child mortality associated with sickle cell anaemia using an indirect approach to overcome the absence of systematic screening at birth.We did a retrospective, multicentre, case-control study in five countries in sub-Saharan Africa (Burkina Faso, Democratic Republic of the Congo, Côte d'Ivoire, Mali, and Senegal). Women with at least one child with a confirmed SS haemoglobin phenotype (sickle cell anaemia) and who had at least three (alive or deceased) children from the same father born more than 5 years ago were recruited at an outpatient consultation in a sickle cell disease care centre. Women who had children without sickle cell disease (control group) were recruited from the same area, with inclusion criteria of being a neighbour or relative of one of the mothers included in the study who had a child with sickle cell anaemia, having no child or other first-degree relative with major sickle cell syndrome, having at least three children (alive or deceased) born more than 5 years ago, and having a confirmed haemoglobin AA phenotype. During the mothers' interview, we collected data concerning the mortality of siblings from the same father of a child with sickle cell anaemia and characteristics of the family, such as age at the time of the survey and the level of education of both parents. Mortality rates were calculated for children younger than 1, 5, and 10 years using the Kaplan-Meier method after excluding the index children. We assumed, as per Mendel law, that in families who have a child with sickle cell anaemia and healthy heterozygous parents, 25% of children born on average have sickle cell anaemia. A multivariate Cox model was used to describe socioeconomic and geographical factors associated with mortality.Between Sept 1, 2017, and Nov 30, 2020, 1563 women who had at least one child with sickle cell anaemia and 4972 women from the same neighbourhood who had children without sickle cell disease were assessed for eligibility. Of 1563 women, 248 were excluded because the genotype of the index child was SC or S β-thalassaemia. 1315 families with cases of sickle cell anaemia and 1243 control families were included in the study. The median age of children (alive) was 14 years (IQR 8-20) in control families and 13 years (8-19) in families with cases of sickle cell anaemia. 5532 [50·6%] of 10 924 children were male. Mortality rates were 15·3% (95% CI 13·3-17·3) for children with sickle cell anaemia younger than 1 year, 36·4% (33·4-39·4) for those younger than 5 years, and 43·3% (39·3-47·3) for those younger than 10 years. Multivariate Cox survival analysis showed that belonging to a family with sickle cell anaemia (hazard ratio [HR] 2·23, 95% CI 1·96-2·54), living in the Democratic Republic of the Congo (HR 1·64, 1·34-2·01), having an older parent (father or mother age had similar effect; HR 1·12, 1·05-1·19 per 10 years of age), or a significantly higher global Multidimensional Poverty Index (HR 1·09, 1·03-1·14), independently increased the risk of mortality. Whereas, living in Senegal (HR 0·70, 95% CI 0·57-0·86) or having a mother with higher education (high school HR 0·66, 0·55-0·80 or advanced HR 0·41, 0·28-0·61) independently decreased the risk of mortality.Although higher than in high-income countries and affected by non-specific socioeconomic factors, the estimated mortality in children with sickle cell anaemia living in sub-Saharan African cities was substantially lower than previous estimates, suggesting an improvement of sickle cell anaemia care in this setting.Fondation Pierre Fabre.For the French translation of the abstract see Supplementary Materials section.

The high frequency of bacterial infections represents a major threat to public health. In developing countries, they are still responsible for significant morbidity and mortality in pediatric populations with sickle cell disease, particularly in children under 5 years of age. Indeed, they have an increased susceptibility to bacterial infections due to their immune deficiency. This susceptibility is even greater for pneumococcal and salmonella infections. In addition, the underdevelopment of some countries and socio-economic factors increases this condition. This review examines the common and specific factors leading to infections in people with sickle cell disease in different types of developed and undeveloped countries. The threat of bacterial infections, particularly those caused by S. pneumoniae and Salmonella, is of increasing concern due to the rise in bacterial resistance to antibiotics. In light of this disturbing data, new strategies to control and prevent these infections are needed. Solutions could be systematic penicillin therapy, vaccinations, and probabilistic antibiotic therapy protocols.

Background: Pulmonary complications of sickle cell disease (SCD) are diverse and encompass acute and chronic disease. The understanding of the natural history of pulmonary complications of SCD is limited, no specific therapies exist, and these complications are a primary cause of morbidity and mortality. Methods: We gathered a multidisciplinary group of pediatric and adult hematologists, pulmonologists, and emergency medicine physicians with expertise in SCD-related lung disease along with an SCD patient advocate for an American Thoracic Society–sponsored workshop to review the literature and identify key unanswered clinical and research questions. Participants were divided into four subcommittees on the basis of expertise: 1) acute chest syndrome, 2) lower airways disease and pulmonary function, 3) sleep-disordered breathing and hypoxia, and 4) pulmonary vascular complications of SCD. Before the workshop, a comprehensive literature review of each subtopic was conducted. Clinically important questions were developed after literature review and were finalized by group discussion and consensus. Results: Current knowledge is based on small, predominantly observational studies, few multicenter longitudinal studies, and even fewer high-quality interventional trials specifically targeting the pulmonary complications of SCD. Each subcommittee identified the three or four most important unanswered questions in their topic area for researchers to direct the next steps of clinical investigation. Conclusions: Important and clinically relevant questions regarding sickle cell lung disease remain unanswered. High-quality, multicenter, longitudinal studies and randomized clinical trials designed and implemented by teams of multidisciplinary clinician-investigators are needed to improve the care of individuals with SCD.

The burden of severe bacterial and malaria infections in children with SCD has been reduced through the use of prophylactic antibiotics and chemoprevention with Sulfadoxine-pyrimethamine. However, such therapies have the potential to promote bacterial and parasitic resistance. To our knowledge, no study has been conducted to determine whether systematic use of prophylactic antimicrobials in children with SCD has an impact on resistance patterns in sub-Saharan Africa. The aim of our study was to determine the incidence and the outcome of severe bacterial and P. falciparum infections in this context. 231 children with SCD and new onset fever associated with either acute pneumonia, urinary tract infection, cholecystitis, meningitis, acute osteomyelitis, or P. falciparum infections were entered into the study. The children in the cohort were an average age of 93 months (± 44 months) and were all followed in a referral center in Western Africa. The 231 children represented 36.67% of the patients regularly followed in the center during the study period and included 183 SS, 26 SC, 12 S?°thal, 10 S?+thal. There were 144 boys and 87 girls in the cohort. The incidence of severe bacterial and Plasmodium falciparum infections were lower than those reported in the general pediatric or sickle cell population in the absence of regular follow-up (ie 5.2, 1.4, 1.0 and, 4.1 per 1000 person/month for urinary tract infections, acute pneumonia, bacteremia and P. falciparum malaria respectively). We observed bacterial strains to be mainly in the Enterobacteria family with high levels of antibiotic resistance. No cases of Streptococcus pneumoniae bacteremia were found. Sulfadoxine-pyrimethamine resistance was observed at high levels. In light of these findings, prophylactic antibiotherapy and antimalarial chemoprevention guidelines in sickle-cell children should be revisited in the context of organized SCD care in sub-Saharan Africa.

Introduction: Sickle Cell Disease (SCD) is one of the most common genetic disease worldwide. The Acute Chest Syndrome (ACS) is a leading cause of death for SCD patients. The PRESEV1 study was set to produce a predictive score to assess the risk of an ACS development (Bartolucci et al., 2016). PRESEV2 was an international, multicenter prospective confirmatory study to validate the PRESEV score. This study aims at improving these predictions with the addition of a machine learning (ML) method. Patients and methods: Included patients follow PRESEV1 and PRESEV2 studies 'rules. The dataset thus contains 97 patients who developed an ACS episode (18.3%) against 434 patients who did not (81.7%). To compute the PRESEV score, we firstly used the method developed previously with the following variables as input: leukocytes, reticulocytes, hemoglobin levels and cervical spine pain. This method is based on a decision tree with fixed rules and is referred to as the decision tree method throughout this abstract. Secondly we used a ML method using a combined sampling method named SMOTEENN to balance the data and a C-Support Vector Classification (SVC) with fixed parameters to predict the score. This method produces a probability, with a threshold of 0.2, under which the patient is predicted to declare an ACS. We considered the dataset composed of PRESEV1 dataset and 80 percent of PRESEV2 with a randomly choice. The test dataset is thus composed of the remaining 20 percent of PRESEV2. This technique of random choice allowed us to use a 50-cross-validation and compute with Python an average score and a standard deviation (std). In order to allow comparison of the developed score with or without the addition of the ML method, rates were calculated by adding the weight of ACS representation in the dataset. Results: Among all parameters analyzed, the SVC method considered the following variables for calculation of the score: leukocytes, LDH, urea, reticulocytes and hemoglobin levels. A hundred and two adult patients with a severe VOC requiring hospitalization were included. Out of this pool of patients, 26 (25.5%) were predicted with a low risk of developing an ACS episode (SVC method). Sensibility and specificity were of 94.7% and 26.8%, respectfully. The negative predictive value (NPV) was of 95.8% and the positive predictive value (PPV) of 22.4%. Results are resumed in table 1. When compared to the PRESEV score (decision tree method), 44 patients out of 372 were identified with a low risk score (11.8%), Discussion and Conclusion: While the addition of a ML method did not allow the improvement of the sensibility or the NPV of the PRESEV score, it improved both the specificity and the PPV. The addition of artificial intelligence thus provides a better prediction with a higher percentage of "low-risk" patients. As highlighted in the international PRESEV study, this score could represent a useful tool for physicians in hospital settings, with limited beds. While the PRESEV score could allow a better management of "low risk" patients on one side, the identification of "high-risk" patients could also represent a serious advantage to physicians, as it could improve the feasibility of clinical trials for the prevention of this lethal complication in SCD patients. Disclosures Bartolucci: Innovhem: Other; Novartis: Research Funding; Roche: Consultancy; Bluebird: Consultancy; Emmaus: Consultancy; Bluebird: Research Funding; Addmedica: Research Funding; AGIOS: Consultancy; Fabre Foundation: Research Funding; Novartis: Consultancy; ADDMEDICA: Consultancy; HEMANEXT: Consultancy; GBT: Consultancy.

Introduction Vaso-Occlusive Crisis (VOC), the most common manifestation of sickle cell disease (SCD), is the first cause of death when complicated by an acute chest syndrome (ACS). We previously developed a predictive score for the occurrence of ACS in SCD patients admitted at hospital for a VOC episode (Bartolucci et al., 2016). Two hundred and fifty patients with severe VOC requiring hospitalization were included in the PRESEV I study and 19% developed a secondary ACS within days post-admission (median [IQR] : 3 [2.3]). A multivariate analysis of these data established a predictive score of secondary ACS, with a negative predictive value of 98,9% for the low-level risk. Variables for calculation of the score were: reticulocytes and leucocytes counts, hemoglobin and categorical pain score (spine -pelvis). Interestingly enough, Hydroxyurea treatment did not have any impact on the score. Our goal was to validate this score in a multicenter international study, as it could represent a useful tool for physicians, for improving VOC management, but also be of use for therapeutic trials. Herein, we present results for adults. Methods This international, multicenter, prospective, observational study was performed in thirteen centres, over two continents (Africa and Europe) and five countries (Mali, Togo, England, Belgium and France). Homozygous SCD patients, both adults and children (>2 years), were included, with severe VOC requiring admission at the emergency unit. Severe VOC was defined as pain or tenderness affecting at least one part of the body (e.g. limbs, ribs, sternum, head, spine and/or pelvis) requiring opioids (level 3) and not attributable to other causes. The primary outcome measure was the occurrence of an ACS, defined as an auscultatory abnormality (crepitation or bronchial breathing), or the association of a new radiologic infiltrate and chest pain or decreased breath sounds. Secondary outcome measures were hospital length of stay, morphine consumption, transfusion, hospitalization in intensive care unit and mortality. The following parameters were recorded: temperature, blood pressure, oxygen saturation, respiratory frequency, categorical pain score. Pulmonary auscultation was performed at least once a day by a physician, every day, for the length of stay. The auscultatory abnormality, defined as crepitation, bronchial breathing or decreased breath sounds, was confirmed by a second physician. If a patient was discharged before day 5, follow-up with a phone call and/or a visit, 3 to 7 days after discharge were performed, to prevent complications or ACS. Results Three hundred and seventy-two adult patients with a severe VOC requiring hospitalization were included. Mean age was 29 (±8) years old, sex ratio (female/male) was 1.2. A hundred and ninety-one patients came from Europe and 181 from Africa. Out of the 372 patients, 68 (18.3%) further developed a secondary ACS. Mean day of ACS episode was 3.3 (±1.37). Among the 304 patients who did not develop an ACS episode (81.7%), 41 had a low risk score of developing a secondary ACS (predictive score ≤ 5). Among the 68 patients who developed a secondary ACS, 43 had a high-risk score (≥ 11). Results are shown in table 1. Three deaths were reported, all on African continent (ACS, anemia and end-stage nephropathy). Discussion and Conclusion Our multicenter international study has allowed confirmation of the incidence of patients developing a secondary ACS during a VOC. This study confirms that the PRESEV score could indeed represent a useful tool for physicians, most especially within emergency structures, by identifying patients with a low risk of further developing an ACS with a good NPV and thus, allowing a better management of SCD patients experiencing a VOC. The PRESEV score was successfully put to practice during the Covid-19 pandemic at the referral SCD center of Henri Mondor hospital and identified "low-risk" patients, who benefited from homecare services (DREPADOM). As such, the PRESEV score could represent a significant help when one considers the shortness of bed availabilities in certain circumstances (pandemics, hospital settings in resource-limited countries, etc...). Finally, the identification of "high-risk" patients could also become useful for improving the feasibility of clinical trials for the prevention of ACS. Disclosures Arlet: Novartis: Consultancy, Honoraria. Bartolucci:Novartis: Research Funding; Emmaus: Consultancy; Addmedica: Research Funding; Fabre Foundation: Research Funding; ADDMEDICA: Consultancy; HEMANEXT: Consultancy; AGIOS: Consultancy; Roche: Consultancy; Bluebird: Consultancy; Bluebird: Research Funding; Innovhem: Other; GBT: Consultancy; Novartis: Consultancy.

Background Sickle cell disease (SCD) accounts for 5% of mortality in African children aged

Growth failure (GF) in children with sickle cell disease (SCD) tends to decline in high-income countries, but data are lacking in sub-Saharan Africa. We performed a cross-sectional study nested in the CADRE (Cœur, Arteres et DREpanocytose) cohort in Mali, Senegal, Cameroon, Gabon and the Ivory Coast. SCD patients and healthy controls aged 5-21 years old were recruited (n = 2583). Frequency of GF, defined as a height, weight or body mass index below the 5th percentile on World health Organization growth charts, was calculated. We assessed associations between GF and SCD phenotypic group, clinical and biological characteristics and history of SCD-related complications. GF was diagnosed in 51% of HbSS, 58% of HbSβ0 , 44% of HbSC, 38% of HbSβ+ patients and 32% of controls. GF in patients was positively associated with parents' lower education level, male sex, age 12-14 years, lower blood pressure, HbSS or HbSβ0 phenotypes, icterus, lower haemoglobin level, higher leucocyte count and microalbuminuria. No association was found between GF and clinical SCD-related complications. In sub-Saharan Africa, GF is still frequent in children with SCD, especially in males and during adolescence. GF is associated with haemolysis and microalbuminuria, but not with the history of SCD-related clinical complications.

Sickle hemoglobin trait protects against severe and uncomplicated Plasmodium falciparum malaria [1–3], but at a cost, in that hemoglobin (Hb) SS disease is associated with significant morbidity and mortality [4]. In contrast, hemoglobin C trait, which is primarily found in West Africa [5], appears to offer protection against severe malaria [6], but hemoglobin CC disease does not have the degree of morbidity and mortality associated with hemoglobin SS disease [7]. Early work demonstrating the protective effects of hemoglobin C trait against severe malaria involved studies of the predominantly Dogon population in east-central Mali [8]. Subsequently, a case-control study in Burkina Faso found that hemoglobin C trait may be protective against both severe and uncomplicated forms of malaria in children [9]. In contrast, a recent longitudinal study of children less than 2 years of age in Ghana found that hemoglobin S trait protected against uncomplicated malaria and anemia (hemoglobin

Resume La vasculopathie drepanocytaire expose a un risque eleve d’accident vasculaire cerebral (AVC) pouvant entrainer le deces ou un handicap important. Le doppler transcrânien (DTC) permet d’identifier les drepanocytaires a risque de cette complication majeure de la maladie, afin de reduire par un traitement approprie, la morbidite et la mortalite qu’elle peut engendrer. De 2008 a 2013, nous avons effectue un depistage systematique de la vasculopathie cerebrale par DTC chez 572 drepanocytaires, âges de 1 a 17 ans et repartis entre 375 sujets SS, 144 SC, 26 S/β 0 et 27 S/β + thalassemiques. Apres exclusion de 30 cas de DTC incomplets et d’un cas de DTC pathologique observe dans un contexte de defaillance multiviscerale, nous avons constate un resultat pathologique ou limite chez 18 % des 541 enfants examines a l’etat basal. Les prevalences de DTC pathologique les plus elevees ont ete observees chez les homozygotes SS (8,1 %). Aucun cas de DTC anormal n’a ete observe chez les enfants S/β + thalassemiques. Les taux d’hemoglobine (Hb) etaient significativement plus bas en cas de resultat au DTC limite ou pathologique. Parmi 68 enfants dont les resultats etaient limites, 9 (13 %) ont presente des resultats pathologiques au bout d’un an de suivi. Dans ce groupe, une diminution ou une augmentation du taux d’Hb de base etait predictive d’un resultat limite ou pathologique au controle. Les enfants dont le resultat etait pathologique ont beneficie, autant que possible, de transfusions ou d’echanges transfusionnels mensuels. Un cas d’AVC transitoire sans signe neurologique, dans un contexte de deglobulisation massive par paludisme et un deces ont ete observes. Ces resultats soulignent la necessite de la pratique systematique du DTC au cours du suivi de l’enfant drepanocytaire et l’urgence de la mise en œuvre de programmes de transfusions chroniques adaptes aux contextes de difficultes d’acces a des transfusions regulieres et securisees.

The hyperhemolysis paradigm that describes overlapping "hyperhemolytic-endothelial dysfunction" and "high hemoglobin-hyperviscous" subphenotypes of sickle cell disease (SCD) patients is based on North American studies. We performed a transversal study nested in the CADRE cohort to analyze the association between steady-state hemolysis and vascular complications of SCD among sub-Saharan African patients. In Mali, Cameroon, and Ivory Coast, 2407 SCD patients (1751 SS or sickle β-zero-thalassemia [Sβ0], 495 SC, and 161 sickle β+-thalassemia [Sβ+]), aged 3 years old and over, were included at steady state. Relative hemolytic intensity was estimated from a composite index derived from principal component analysis, which included bilirubin levels or clinical icterus, and lactate dehydrogenase levels. We assessed vascular complications (elevated tricuspid regurgitant jet velocity [TRV], microalbuminuria, leg ulcers, priapism, stroke, and osteonecrosis) by clinical examination, laboratory tests, and echocardiography. After adjustment for age, sex, country, and SCD phenotype, a low hemoglobin level was significantly associated with TRV and microalbuminuria in the whole population and with leg ulcers in SS-Sβ0 adults. A high hemolysis index was associated with microalbuminuria in the whole population and with elevated TRV, microalbuminuria, and leg ulcers in SS-Sβ0 adults, but these associations were no longer significant after adjustment for hemoglobin level. In conclusion, severe anemia at steady state in SCD patients living in West and Central Africa is associated with elevated TRV, microalbuminuria, and leg ulcers, but these vascular complications are not independently associated with indirect markers of increased hemolysis. Other mechanisms leading to anemia, including malnutrition and infectious diseases, may also play a role in the development of SCD vasculopathy.

Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the history of the disease in this continent remains largely unknown. SCD is characterized by chronic hemolytic anemia, acute-vaso occlusive events and progressive vascular organ damage. The CADRE study is a large cohort of SCD patients in five countries of West and Central Africa aiming at studying SCD-related vascular complications. The inclusion data of this study did not match the hyper-hemolysis paradigm (Dubert et al. Blood 2018), but several methodological limitations were raised, including probable mortality bias and questionable reliability of classical hemolysis markers in Africa. For the 5-year follow-up of the CADRE study, we designed a case-control study nested in the cohort, based on extreme phenotypes, to look for new markers of vasculopathy, including new markers of hemolysis. Patients and Methods SS adult patients of the CADRE cohort were selected in the centres of Dakar (Senegal) and Bamako (Mali), depending on the presence of none or at least one of the following complications at inclusion: tricuspid regurgitant jet velocity (TRJV)>2,5 m/s (which may indicate pulmonary hypertension), albuminuria/creatininuria>100 mg/g, leg ulcer, priapism, aseptic osteonecrosis and retinopathy. We chose the youngest patients with a vascular complication and the oldest without any complication. Overall, 6 groups of 40 SS patients with extreme phenotypes were constituted (20 in each centre). Patients were called for a specific visit and investigated at steady state. Besides clinical examination, usual laboratory blood tests and albuminuria measure, additional plasma and saliva samples were collected. A trained investigator isolated microparticles immediately after blood sampling by successive centrifugations, measured blood and plasma viscosities and assessed microcirculation function using peripheral arterial tonometry. A cardio-echography was performed by a trained cardiologist. Plasma samples were stored at -80 °C and shipped to Paris. High technology tests were performed in Paris, including blood cell derived microparticles, free hemoglobin, inflammatory cytokines, neutrophile extracellular trap (NETs). Using saliva DNA, we also genotyped the known SCD genetic modifiers and new candidate genes implicated in the catabolism of hemoglobin. Potential associations between those markers, usual hematological parameters, and the vascular complications were assessed statistically . Results We recalled 240 selected patients 5 years after their first visit: 38 could not be retrieved, 21 had deceased, 62 had at least one new complication, and only 15 still had no complication. Therefore, we selected 56 more patients to obtain at least 30 patients in each group. 237 SS adults were eventually investigated and the plasma samples of 232 SS patients were analyzable in Paris (106 from Bamako and 126 from Dakar). In these patients, at a mean age of 29 years (+/- 11), high TRJV was present in 58, macroalbuminuria in 33, leg ulcers in 36, priapism in 43, aseptic osteonecrosis in 45 and retinopathy in 31 whereas 28 had no vascular complication. A principal component analysis found no cluster of complications. Among patients with one "hyper-viscous" complication (retinopathy or osteonecrosis) or more, 78% also had at least one "hyper-haemolytic" complication (high TRJV, albuminuria, leg ulcer or priapism) and 49% of patients with a "hyper-haemolytic" complication also had a "hyper-viscous" complication. The microvascular function was not associated with any of the complications, whereas higher blood viscosity was associated with retinopathy. The results of the associations between the vascular complications and specific biological tests are presented in other publications. Conclusion This study illustrates the feasibility of high-technology experiments in SCD patients living in sub-Saharan Africa, but was particularly challenging because of the difficulty to prepare, store and transport frozen plasma samples. Moreover, in agreement with previously published data from the CADRE study, we found that the dichotomization of vascular complications into hyperhemolytic and hyperviscous subgroups is not clinically relevant in Africa. Other simple predictive markers of vascular complication are needed to optimize the follow-up of African patients with SCD. Disclosures No relevant conflicts of interest to declare.

Introduction Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the history of the disease on this continent remains largely unknown. SCD is characterized by the association of chronic hemolytic anemia with episodes of acute vaso-occlusive events and progressive vascular organ damage. Several pathophysiological pathways in SCD result in the activation of circulating blood cells and the release of microparticles (MPs). In the present study, we investigated cell-derived MPs in patients with SCD living in Africa and analyzed their relationship with clinical complications. Patients and Methods This cross-sectional case-control study is nested in the CADRE cohort (clinical trials.gov identifier NCTO3114137). We included 232 SS adults in two African centers: Bamako (Mali) and Dakar (Senegal). Patients were chosen depending on the absence or the presence of at least one of the following complications: tricuspid regurgitant jet velocity (TRJV) >3 m/s (which may indicate pulmonary hypertension), macroalbuminuria, leg ulcer, priapism, aseptic osteonecrosis, and retinopathy. Overall, 7 groups of 40 SS patients were constituted (20 in each center). Patients were investigated at steady state (i.e.,at least 15 days after a vaso-occlusive crisis, 8 days after fever or infectious disease, and 3 months after a transfusion). MPs were isolated in the African centers immediately after blood sampling by successive centrifugations at increasing speed: 2,500g x2 and 21,000g x2. MPs pellets were stored at -80 °C. The cellular origin of the MPs, erythrocyte, reticulocyte, endothelial, platelet, and leucocyte, was determined using antibodies directed against CD235a, CD71, CD106, CD41, and CD45, respectively, at the National Institute of Blood TransfusioninParis. To maintain the background at an acceptable level, events of 0.16 µm size were excluded (Fig 1A). Only MPs positive for Annexin V and the cell-type-specific labelling were retained. Potential associations between cell-derived MPs, hematological parameters, and vascular complications were assessed using logistic regression with adjustment for age, sex and country. Results The MP pellets of 106 SS patients from Bamako and 126 from Dakar were analyzed in Paris. In these patients, at a mean age of 29 years (+/- 11), high TRJV was present in 64, microalbuminuria in 84, leg ulcers in 33, priapism in 43, aseptic osteonecrosis in 45, and retinopathy in 31 patients whereas 28 patients had no complication at the time of sampling. As a typical result, Fig 1B shows erythrocyte-derived MPs labelled by Annexin V and CD235a (quarter Q2). The MPs distribution was as follows: erythroid 52% [reticulocytes (CD235a+CD71+) 14%, erythrocytes (CD235a+ CD71-) 38%], leukocyte 18%, platelet 21%, and endothelial 9%. Neither erythrocyte- nor reticulocyte-derived MPs significantly correlated with hemolysis markers (LDH, unconjugated bilirubin or reticulocytes) or hemoglobin levels. Erythrocyte- and reticulocyte-derived MPs were significantly lower in patients with retinopathy (OR=0.48, p=0.003 and OR=0.68, p=0.005, respectively). Reticulocyte-derived MPs were negatively associated with a history of priapism (OR=0.76, p=0.020) and positively associated with a history of leg ulcers (OR=1.23, p=0.040). No correlation was found between MPs of other cellular origin and chronic complications, except for a negative association between endothelial-derived MPs and priapism (OR=0.76, p=0.036). Conclusion In our African patients with SCD, erythroid-derived MPs, although recognized cellular products of hemolysis, were not associated with other markers of hemolysis. We hypothesize that erythroid MPs are not only derived from hemolysis but also probably from the sickling process in this population. They were negatively associated with retinopathy and priapism and positively associated with leg ulcers, but not with the other complications classically associated with the hyperhemolytic sub-phenotype. The search for pertinent biomarkers of SCD complications in Africa is an essential challenge. To our best knowledge, this report is the first illustrating the feasibility of high-technology experiments in an African context. Disclosures No relevant conflicts of interest to declare.

Red cell transfusion is one of the main treatments in sickle cell disease. However there are potential risks of blood transfusions. In order to propose strategies to improve blood safety in sickle cell disease in Mali, we conducted a prospective study of 133 patients with sickle cell anemia recruited at the sickle cell disease research and control center of Bamako, November 2010 to October 2011. The study aimed to determine the prevalence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections by serum screening and the frequency of red cell alloimmunization before and after blood transfusion. The diagnosis of sickle cell syndrome was made by HPLC, the detection of markers of viral infection was performed by ELISA, and the diagnosis of alloimmunization was conducted by the Indirect Coombs test. Prevalence of viral infections observed at the time of enrolment of patients in the study was 1%, 3% and 1% respectively for HIV, HBV and HCV. Three cases of seroconversion after blood transfusion were detected, including one for HIV, one for HBV and one another for HCV in sickle cell anemia patients. All these patients had received blood from occasional donors. The red cell alloimmunization was observed in 4.4% of patients. All antibodies belonged to Rh system only. Blood transfusion safety in sickle cell anemia patients in Mali should be improved by the introduction of at least the technique for detecting the viral genome in the panel of screening tests and a policy of transfusions of blood units only from regular blood donors.

The goal of this study was CGH array profiling of breast cancer from Malian women in order to define differences with those from USA. CGH array was performed in 28 samples, 17 with a triple negative phenotype. The profiles were compared to those of 106 tumors from USA. 6 chromosomal regions (6p21, 9q34, 11q13, 12q24, 17q25 and 22q12.1-22q13.1) were identified with a significant higher rate of copy number alterations. These regions contain several genes of interest including BCR. FISH and IHC confirmed that BCR was amplified and overexpressed particularly in triple negative tumors. Finally, 5 regions presented a high level of amplification in two or more samples, including 2 regions located between 9p22.3-9p23 and 9p23-9p24.1. This study confirms that breast cancers from African women present biological differences with those from USA. Larger studies are needed to go further in the identification of therapeutic targets that would be specific to African women.

Resume Dans les contextes ou la biologie moleculaire n’est pas accessible, la caracterisation des syndromes thalassemiques se fait a partir des donnees de l’hemogramme, du bilan martial et du typage des hemoglobines. Le typage des hemoglobines fait alors recours a la technique d’electrophorese a pH alcalin. Cette technique ne permet pas de separer et de quantifier les hemoglobines C et A2. La separation des hemoglobines C et A2, suivie de leur quantification, est rendue possible depuis l’avenement des electrophoreses en capillaire de zone. Pour caracteriser les syndromes thalassemiques dans le contexte malien caracterise par une grande frequence du gene de l’hemoglobine C, nous avons decide de determiner les valeurs de reference de l’hemoglobine A2 dans une population du district de Bamako au Mali. L’etude a ete conduite aupres de 41 volontaires âges de 6 mois a 45 ans tous sexes confondus. L’electrophorese de l’hemoglobine a ete faite selon la technique en capillaire de zone a l’aide d’un automate Capillarys 2 des laboratoires Sebia. Les parametres biologiques consideres pour l’etude ont ete les constantes erythrocytaires ; les taux des reticulocytes, de la ferritine serique et des fractions hemoglobiniques. La saisie et l’analyse des donnees ont ete faites sur SPSS 12.0. Le test d’ANOVA a ete utilise pour la comparaison des valeurs moyennes et des medianes avec un seuil de signification fixe a un p ≤ 0,05. Les percentiles 2,5 et 97,5 de l’hemoglobine A2 ont ete calcules. Les valeurs de reference de l’hemoglobine A2 observees dans la population etudiee etaient comprises entre 2,50 et 3,68% (moyenne = 2,98 ± 0,23). Ces valeurs ne differaient pas selon l’âge ou le sexe des participants a l’etude ; elles differaient en revanche de celles publiees par d’autres auteurs chez des populations differentes de la notre. A defaut de biologie moleculaire, ces resultats permettent une meilleure caracterisation des syndromes thalassemiques dans notre contexte et soulignent l’interet de determiner systematiquement les valeurs de reference des fractions d’hemoglobine de chaque population.

Chronic kidney disease is one of the leading causes of mortality in patients with sickle cell disease. However, it has been almost exclusively studied in patients with the SS phenotype and in high-income countries, despite more than 80% of patients living in Africa. We looked for the determinants of glomerulopathy in a multinational cohort of patients with sickle cell disease of different phenotypes in sub-Saharan Africa.In the CADRE cohort, we prospectively included patients 3 years and older with sickle cell disease of all haemoglobin phenotypes in Cameroon, Côte d'Ivoire, Mali, and Senegal. All individuals were assessed at steady state. The main outcome of interest was albuminuria defined as a urine albumin-to-creatinine ratio of greater than 30 mg/g. We investigated the clinical and biological determinants (including haemolysis markers) of albuminuria in two main phenotype groups (SS and Sβ(0); SC and Sβ(+)) with further stratification by age and country.The study is ongoing because of follow-up. 2582 patients with sickle cell disease were included (1776 SS, 136 Sβ(0), 511 SC, and 159 Sβ(+)). 644 patients with the SS and Sβ(0) phenotypes (33·7%, 95% CI 31·6-35·8) and 110 with the SC and Sβ(+) phenotypes (16·4%, 13·6-19·2) had albuminuria. In the SS and Sβ(0) group, albuminuria was detected in 144 (27%) of 527 children younger than 10 years and its frequency increased with age (29 [48%] of 60 patients aged40 years). Multivariable analysis showed that albuminuria was associated with age (odds ratio 1·43, 95% CI 1·20-1·71; p0·0001), female sex (1·35, 1·02-1·82; p=0·045), low haemoglobin (0·79, 0·66-0·93; p=0·006), high lactate dehydrogenase concentrations (1·33, 1·14-1·58; p=0·0009), and, using Côte d'Ivoire as the reference, Mali (2·49, 1·64-3·79; p=0·042) and Cameroon (1·59, 1·01-2·51; p=0·0007) in patients with the SS and Sβ(0) phenotypes. The magnitude of the association of albuminuria with haemoglobin and lactate dehydrogenase concentrations increased with age. In the SC and Sβ(+) patients, only low haemoglobin (0·69, 0·48-0·97; p=0·029), high blood pressure (1·63, 1·17-2·27; p=0·0017), and Mali (3·75, 1·75-8·04; p0·0001) were associated with albuminuria.Hyperhaemolysis is associated with albuminuria, with an age-dependent effect, in the SS and Sβ(0) phenotypes only, suggesting a different pathological mechanism for glomerular disease in the patients with SC and Sβ(+) phenotypes. However, both phenotypes are associated with a high prevalence of albuminuria in childhood. Therefore, screening for albuminuria is advised in African children with sickle cell disease to detect early renal damage.Paris Cité Sorbonne University (GrEX project) and Cardiology and Development.

Background The safety and immunogenicity of PfAMA1, adjuvanted with Alhydrogel® was assessed in malaria–experienced Malian adults. The malaria vaccine, PfAMA1-FVO [25-545] is a recombinant protein Pichia pastoris-expressed AMA-1 from Plasmodium falciparum FVO clone adsorbed to Alhydrogel®, the control vaccine was tetanus toxoid produced from formaldehyde detoxified and purified tetanus toxin. Methods A double blind randomized controlled phase 1 study enrolled and followed 40 healthy adults aged 18–55 years in Bandiagara, Mali, West Africa, a rural setting with intense seasonal transmission of P. falciparum malaria. Volunteers were randomized to receive either 50 µg of malaria vaccine or the control vaccine. Three doses of vaccine were given on Days 0, 28 and 56, and participants were followed for 1 year. Solicited symptoms were assessed for seven days and unsolicited symptoms for 28 days after each vaccination. Serious adverse events were assessed throughout the study. The titres of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed. Results Commonest local solicited adverse events were the injection site pain and swelling more frequent in the PfAMA1 group. No vaccine related serious adverse events were reported. A significant 3.5-fold increase of anti-AMA-1 IgG antibodies was observed in malaria vaccine recipients four weeks after the third immunization compared to the control group. Conclusion The PfAMA1 showed a good safety profile. Most adverse events reported were of mild to moderate intensity. In addition, the vaccine induced a significant though short-lived increase in the anti-AMA1 IgG titres. Registered on www.clinicaltrials.gov with the number NCT00431808 Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1466-4) contains supplementary material, which is available to authorized users.

Background: Although a blood genetic disease, sickle cell disease (SCD) leads to a chronic vasculopathy with multiple organ involvement. We assessed arterial stiffness in SCD patients and looked for associations between arterial stiffness and SCD-related vascular complications. Methods: The CADRE (Coeur Artères et Drepanocytose, ie, Heart Arteries and Sickle Cell Disease) study prospectively recruited pediatric and adult SCD patients and healthy controls in Cameroon, Ivory Coast, Gabon, Mali, and Senegal. Patients underwent clinical examination, routine laboratory tests (complete blood count, serum creatinine level), urine albumin/creatinine ratio measure, and a measure of carotid-femoral pulse wave velocity (cf-PWV) and augmentation index (AI) at a steady state. The clinical and biological correlates of cf-PWV and AI were investigated by using a multivariable multilevel linear regression analysis with individuals nested in families further nested in countries. Results: Included were 3627 patients with SCD and 943 controls. Mean cf-PWV was lower in SCD patients (7.5±2.0 m/s) than in controls (9.1±2.4 m/s, P 0 than in SC-Sβ + phenotypes. AI, corrected for heart rate, increased more rapidly with age in SCD patients and was higher in SCD than in control adults. cf-PWV and AI were independently associated with age, sex, height, heart rate, mean blood pressure, hemoglobin level, country, and hemoglobin phenotype. After adjustment for these correlates, cf-PWV and AI were associated with the glomerular filtration rate and osteonecrosis. AI was also associated with stroke, pulmonary hypertension, and priapism, and cf-PWV was associated with microalbuminuria. Conclusions: PWV and AI are deeply modified in SCD patients in comparison with healthy controls. These changes are independently associated with a lower blood pressure and a higher heart rate but also with the hemoglobin phenotype. Moreover, PWV and AI are associated with several SCD clinical complications. Their prognostic value will be assessed at follow-up of the patients.

Human parvovirus B19 (HP-19) is the only Parvoviridae known to be pathogenic in human. Studies of HP-19 infection and its associated life-threatening complications in sickle cell anemia patients have been reported in Europe and the US. These results justify the development of HP-B19 prevention and strategies to reduce the incidence of severe and life-threatening complications associated with the infection in patients with sickle cell anemia, particularly in sub-Saharan Africa where the sickle cell anemia burden is high. In light of these considerations, we conducted a case-control study including 163 patients with sickle cell anemia and 163 controls. HP-B19 diagnosis was based on the detection of IgG and IgM antibodies specific for HP-B19 using commercially available enzyme immunoassays. Anti-human parvovirus B19 IgG antibodies were found in 105 of 193 (64.8%) patients vs 79 of 193 controls (48.4%). IgM antibodies were found at a higher frequency in sickle cell anemia patients than in controls. This higher frequency was found to be age-dependent. However, the reticulocyte count showed no significant decrease in Malian patients with sickle cell anemia. Further studies are needed to better characterize the implication of HP-B19 infection in sickle cell anemia mortality and morbidity and to develop preventive strategies and efficient management of the resulting complications.

Breast cancer is the second most frequent cancer in Sub-Saharan African women with an incidence of 15-53 per 100,000 women. Using PubMed, we reviewed all the articles published on this topic between 1989 and 2009. Breast cancer is usually diagnosed in women younger than in developed countries (mean age: 42-53 years), with later stages (III or IV, i.e. with axillary nodes and distant metastases). Reported tumors are mostly invasive ductal carcinomas with aggressive characteristics: grade III histoprognosis, absence of hormonal receptors or HER2 expression. According to the new breast cancer classification, nearly half of these tumors should be classified as triple negative. However, studies are rare and require confirmation. In conclusion, data on epidemiology and biology of breast cancer in Sub-Saharan African women are still scarce and need more extensive studies. In these countries, the pattern of breast cancer will likely change in the future, according to the evolution of lifestyle namely urbanisation. There is a great need for commitment of research and clinical resources in Sub-Saharan Africa in order to develop specific strategies.

Resume Cette etude prospective conduite sur une periode de neuf mois avait pour objectif de determiner la frequence de l’allo-immunisation erythrocytaire chez les malades polytransfuses du service d’hematologie-oncologie medicale et de l’unite d’hemodialyse du service de nephrologie du CHU du Point-G. Le depistage et l’identification des anticorps ont ete realises par des techniques en gel filtration en Coombs indirect et en milieu enzymatique. Il n’a pas ete effectue de reactions en milieu salin. Au total 78 patients ont ete enroles dans les deux services de recrutement. L’âge moyen des malades etait de 36,78 ± 14,73 ans avec des extremes de 11 et 77 ans. Le sexe ratio etait de 1,11 en faveur des femmes. La moyenne de poches transfusees etait de 12,21 ± 9,99 poches avec des extremes de 4 et 45 poches. Les phenotypes Rhesus Dccee, DccEe et DCcee ont ete les plus rencontres, avec des frequences respectives de 67,9, 15,4 et 10,3 % ; et l’antigene K a ete retrouve a une frequence de 1,28 %. La frequence de l’allo-immunisation etait de 10,3 % dans notre serie. Il n’y avait pas de difference significative entre les deux services de recrutement. Toutes les agglutinines depistees etaient des anticorps chauds appartenant au systeme Rhesus : anti-E (7,7 %), anti-C (1,3 %) et anti-D (1,3 %). Seul l’anti-E etait present chez les patients souffrant d’une affection hematologique. Nous n’avons pas trouve de liaison statistiquement significative entre le sexe, l’âge, le nombre de poches transfusees et la positivite de la recherche d’agglutinines irregulieres (RAI). Nous concluons que la frequence de l’allo-immunisation post-transfusionnelle est elevee chez les malades polytransfuses au Mali. La RAI systematique chez ces malades et la selection de concentres de globules rouges phenotypes dans les systemes Rhesus/Kell permettraient une securite transfusionnelle optimale.

Introduction Growth failure has been a well-known complication of sickle cell disease (SCD) since the 70s. More recent studies show that the proportion of underweight children with SCD has decreased significantly, thanks to modern treatments (in particular hydroxyurea and iterative transfusions), with a tendency towards overweight and even obesity. However, most studies have been carried out in high-income countries, while 80% of the affected children are born in sub-Saharan Africa, where the environment and the medical care are entirely different. We carried out a case-control study nested in a multinational African cohort to study the growth of sickle cell children and the possible factors influencing growth failure. Methods We performed a case-control transversal study nested in the CADRE cohort that includes SCD patients from five African countries: Cameroon, Gabon, Côte d'Ivoire, Mali and Senegal. All children aged 5 to 21 years-old from this cohort were included in our study. Healthy controls were recruited among the patients' siblings or the children of health workers from each center. The main parameters studied were: medical history, height, weight, blood pressure; hemoglobin phenotype (SS, Sβ0, SC or Sβ +); complete blood count; hemolysis markers (LDH and bilirubin); microalbuminuria; echocardiographic parameters. The primary endpoint was growth failure, defined as a weight, height or BMI below the 5th percentile of the WHO growth charts. We described the frequency of growth failure according to hemoglobin phenotype, age and sex. Then we assessed by multivariate logistic regression in two SCD phenotypic groups (SS or Sβ0 and SC or Sβ +) the association between growth failure and the biological characteristics or the history of SCD-related complications. Results 2296 patients (1799 SS, 114 Sβ0, 287 SC, 96 Sβ+ patients and 287 controls were enrolled in Cameroon (n=735), Ivory Coast (n=380), Gabon (n=298), Mali (n=589) and Senegal (n=581). Overall, 48% of the patients were male and their median [interquartile range] age was 12 [8-16] years-old. Growth failure was diagnosed in 51% of SS, 58% of Sβ0, 44% of SC, 38% of Sβ+patients and 32% of controls, with deeper underweight than linear growth retardation. Beyond the age of 18, the mean BMI of SCD patients was again similar to that of controls in girls, but remained lower in boys, whereas the mean height was similar to that of controls regardless of sex (Figures a to d). Growth failure was more frequent in boys than in girls and maximal between 13 and 16 years-old (Figures e and f). In univariate analysis, the prevalence of growth retardation was associated with the country (highest in Senegal, lowest in Cameroon), age, male sex, hemoglobin phenotype, levels of anemia and hemolysis (p Conclusion In sub-Saharan Africa, growth failure occurs in more than half of the SCD children and concerns weight more than height. Its prevalence is particularly high in SS or Sβ0 patients and during adolescence, due to pubertal delay. Growth failure in SCD children is associated with male sex, anemia and high hemolysis markers independently of the hemoglobin phenotype, but is not independently associated with the occurrence of acute or chronic vascular complications of sickle cell disease, apart from microalbuminuria. However, the long-term consequences of growth failure in sickle cell disease should be evaluated in a longitudinal study. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The prevalence of sickle cell disease is extremely high in Africa, where 150,000 to 300,000 homozygous individuals are born every year. Africans were well aware of the disease before its first description in America. Spectacular progress has been made in our knowledge of the pathophysiology of SCD, and patient management has vastly improved in northern countries where the prevalence of the disease is low. We describe the current situation of sickle cell disease in Africa and propose strategies for improving disease prevention and patient management.

Severe bacterial infections are the major causes of morbidity and mortality in sickle cell anemia (SCA) but are poorly explained. The distribution of a bi-allelic polymorphism (Arg107Gly) of human leukocyte antigen-E (HLA-E) locus was investigated in 144 SCA patients, most of whom originated from from sub-Saharan Africa. Among them, 73 presented with at least one severe bacterial infection, whereas 71 did not. The HLA-E*0101/E*0101 genotype was more frequent among the group with infections than their counterparts (47% vs 21%; p corrected = 0.003). This genetic association is of relevance, given the emerging evidence for the involvement of HLA-E molecules in host response to pathogens.

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.

Monocyte dysfunction in filarial infection has been proposed as one mechanism underlying the diminished antigen-specific T-cell response seen in patent lymphatic filariasis. Cytokine/chemokine production and gene expression in monocytes from filaria-infected patients and uninfected healthy donors were assessed unstimulated and in response to stimulation withStaphylococcus aureusCowan I bacteria plus gamma interferon both before and 8 months following treatment. Monocytes from filaria-infected individuals were studded with intracellular microfilarial antigens. Furthermore, monocytes from these individuals were less capable of producing interleukin-8 (IL-8), Exodus II, MIP-1α, MIP-1β, and IL-1α and preferentially expressed genes involved in apoptosis and adhesion compared with monocytes from uninfected donors. Eight months following treatment with a single dose of ivermectin-albendazole, some of these defects were reversed, with monocyte production of IL-8, IL-1α, MIP-1α, and IL-10 being comparable to that seen in the uninfected controls. In addition, a marked increase in mRNA expression of genes associated with protein metabolism, particularly heat shock proteins, was seen compared with pretreatment expression. These data suggest that the function and gene expression of monocytes in filaria-infected patients are altered but that this dysfunction is partially reversible following antifilarial treatment.

The Knops-Helgeson antigen was first reported by Helgeson and coworkers1 in an article describing a weak, antiglobulin-reactive antibody that was found in a 37-year-old Caucasian woman. A family study of the only compatible donor (MH) showed that the antigen was independent from the loci for ABO, MNSs, Rh, Lu, Jkm and Se; hence it was given the name Knops-Helgeson and abbreviated as Kna. A serum sample that reacted with 4.5 percent of Caucasian donors was later reported as being anti-Knb; however, this antigen was not found among African American blood donors.2 During the initial period when the Knops blood group system was being investigated, a number of antibodies with similar characteristics were described and placed in a group known as “high-titer, low avidity” (HTLA) antibodies. The group included Chido, Rodgers, York, Csa, JMH, Knops, McCoy, and Swain-Langley which have now been assigned to separate blood group systems. One of the characteristics of these antibodies was their weak and variable reactivity. Accurate phenotyping of RBCs was difficult and reference laboratories were often unable to duplicate each other's results. The identification of the Knops antigens on complement receptor 1 (CR1) began to explain some of these findings but also revealed the complexity of this system.3,4 Moulds and colleagues5 reported that low expression of RBC-CR1 often resulted in weak or negative Knops typings. Consequently, better methods were needed to correctly identify antigen-positive versus antigen-negative individuals. A concerted effort to identify the molecular basis of the Knops alleles began following the report that CR1 was a ligand in the rosetting of Plasmodium-infected RBCs with noninfected cells.6 Early work by Molthan7 and by Moulds8 indicated that significant differences in several Knops antigen frequencies existed between Caucasian and African American persons. It was postulated that some Knops blood group phenotypes, for example, Sl(a−), might be protective against rosetting and severe malaria.6 Thus, the CR1 gene was systematically investigated to find mutations that might correlate with the blood group antigen specificities. The most common form of the CR1 gene, that is, CR1*1, is composed of 38 exons spanning 133 kb producing a protein of 220 kDa. Large insertions and deletions have given rise to four structural variant genes but these are unrelated to the blood group antigens.9 Approximately 60 amino acids comprise a short consensus repeat (SCR), and seven SCRs are further arranged into four long homologous repeats. Because of this high degree of homology, locating the blood group antigens was not an easy task, but the single-nucleotide changes resulting in the McCa, McCb, Sla, and Vil antigens have now been identified in SCR25.9 The Sla antigen has recently been subdivided10 and, hence, Sla has been renamed Sl1 and Vil has been redesignated as Sl2.11 We now report the molecular identification of the third allelic pair, Kna/Knb, and outline a simple method for genotyping these polymorphisms.

Residents of malaria-endemic areas sometimes spontaneously clear Plasmodium falciparum infection without drug treatment, implying an important role for host factors such as immunity in this clearance. Host factors may also contribute to clearance of parasites resistant to a treatment drug. Chloroquine resistance is caused by point mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene. We investigated the clearance of malaria parasites carrying the key chloroquine resistance-conferring PfCRT mutation K76T in patients treated with chloroquine. We found that the ability to clear these resistant parasites is strongly dependent on age (the best surrogate for protective immunity in endemic areas), suggesting that host immunity plays a critical role in the clearance of resistant P. falciparum infections. Age-adjusted comparison of subjects able to clear resistant parasites and those unable to do so provides a new phenotype for identifying host immune and genetic factors responsible for protective immunity against malaria.

Post-treatment reactions to single-dose ivermectin (200 μg/kg) and albendazole (400 mg) were studied in a filarial endemic region of Mali. The prevalence of Wuchereria bancrofti in this region was 48.3% (69 of 143), and coinfection with Mansonella perstans was common (30 of 40, 75%). Microfilarial levels of M. perstans correlated positively with age (P = 0.006) and with W. bancrofti microfilarial levels (P = 0.006). Forty individuals (28 infected and 12 uninfected) were treated, with mild post-treatment reactions occurring in 35.7% (7 of 28) of the W. bancrofti-infected subjects. Reaction severity correlated with pretreatment W. bancrofti microfilarial levels (P = 0.001). There were no significant differences in the prevalence or severity of post-treatment reactions in those who were co-infected with M. perstans. It is concluded that co-infection with M. perstans does not significantly alter the post-treatment reaction profile to single-dose ivermectin/albendazole in W. bancrofti infection in this community, and that acute post-treatment reactions should not limit patient compliance in community-based programs to eliminate lymphatic filariasis.

Peripheral parasite density of Plasmodium falciparum is used as an indicator of malaria disease severity, but does not quantify central sequestration, which is important in the pathogenesis of severe disease. Malaria pigment, recognizable within the cytoplasm of phagocytic cells by light microscopy may represent a peripheral marker for parasite biomass. One hundred seventy-two index cases of severe malaria and 172 healthy age-, residence-, and ethnicity-matched controls with uncomplicated malaria in Bandiagara, Mali were analyzed prospectively for presence of malaria pigment. The presence of polymorphonuclear cell (PMN) and monocyte pigment was strongly associated with severe disease compared with uncomplicated malaria. Total PMN pigment burden in children with severe malaria was higher in those with cerebral manifestations and with combined cerebral manifestations and severe anemia (hemoglobin 5 g/dL) but was not associated with hyperparasitemia (> 500,000 asexual forms/mm 3 ). Additionally, pigmented PMNs/mm 3 was associated with a fatal outcome in patients with severe malaria. This study validates the presence of malaria pigment in monocytes and neutrophils as a marker for disease severity, and demonstrates that pigmented neutrophils are associated with cerebral malaria and with death in children with severe malaria.

Elucidating the genomic diversity of CD209 gene promoter polymorphisms could assist in clarifying disease pathophysiology as well as contribution to co-morbidities. CD209 gene promoter polymorphisms have been shown to be associated with susceptibility to infection. We hypothesize that CD209 mutant variants occur at a higher frequency among Africans and in sickle cell disease. We analyzed the frequency of the CD209 gene (rs4804803) in healthy control and sickle cell disease (SCD) populations and determined association with disease. We obtained genomic DNA from 145 SCD and 244 control Africans (from Mali), 331 SCD and 379 control African Americans and 159 Caucasians. Comparative analysis among and between groups was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Per ethnic diversification, we found significant disparity in genotypic (23.4% versus 16.9% versus 3.2%) and allelic frequencies (36.1% versus 25.1% versus 11.6%) of the mutant variant of the CD209 (snp 309A/G) gene promoter between Africans, African Americans and Caucasians respectively. Surprisingly, there was a wide disparity in the genotypic and allelic frequencies among African SCD versus healthy controls (10.4% versus 23.4% (genotypes) and 25.2% versus 36.1% (alleles), which is completely absent among African Americans. Comparing SCD groups, there was no difference between Africans and Americans, implying a lack of association between CD209 polymorphisms and sickle cell disease in either population. The higher frequency of CD209 mutant variants in the non-SCD group reveals an impaired capacity to mount an immune response to infectious diseases. We conclude that CD209 polymorphism play a major role in susceptibility to infectious pathogens and could potentially delineate susceptibility to and severity of co-morbidities.

Elucidating the genomic diversity of CD209 gene promoter polymorphism could assist in clarifying disease pathophysiology as well as contribution to co-morbidities. CD209 gene promoter polymorphism has been shown to be associated with susceptibility to infection. We hypothesize that CD209 mutant variants occur at a higher frequency among Africans and in sickle cell disease. We analyzed the frequency of the CD209 gene (rs4804803) in healthy control and sickle cell disease (SCD) populations and determined association with disease. Genomic DNA was extracted from blood samples collected from 145 SCD and 231 control Africans (from Mali), 331 SCD and 379 control African Americans and 159 Caucasians. Comparative analysis among and between groups was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Per ethnic diversification, we found significant disparity in genotypic (23.4% versus 16.9% versus 3.2%) and allelic frequencies (48.7% versus 42.1% versus 19.8%) of the homozygote mutant variant of the CD209 (snp 309A/G) gene promoter between Africans, African Americans and Caucasians respectively. Comparative evaluation between disease and control groups reveal a significant difference in genotypic (10.4% versus 23.4%; p = 0.002) and allelic frequencies (39.7% versus 48.7%; p = 0.02) of the homozygote mutant variant in African SCD and healthy controls respectively, an observation that is completely absent among Americans. Comparing disease groups, we found no difference in the genotypic (p = 0.19) or allelic (p = 0.72) frequencies of CD209 homozygote mutant variant between Africans and Americans with sickle cell disease. The higher frequency of CD209 homozygote mutant variants in the African control group reveals a potential impairment of the capacity to mount an immune response to infectious diseases, and possibly delineate susceptibility to or severity of infectious co-morbidities within and between groups.

Author(s): Diarra, AB; Kouriba, B; Guindo, A; Maiga, AI; Diabate, DT; Douyon, I; Diawara, SI; Toure, BA; Fonkoro, S; Murphy, EL; Diallo, DA

In the late 1990s publications on cohorts of sickle cell disease (SCD) patients, followed since birth, showed that the life expectancy of SCD patients in developed countries could approach that of those without SCD, when managed appropriately. Between 2005 and 2008, SCD was declared as a public health priority issue worldwide. In 2006, the WHO recommended that African states should include the fight against SCD in their health policies. Nevertheless, there are, as of yet, no data on effective strategies to implement SCD control in these countries. This review discusses the stakes and proposes strategies for SCD management and research in sub-Saharan Africa.This work is a review of the recent literature on the burden of SCD in sub-Saharan Africa; on approaches that resulted in improved survival and comfort for SCD patients in developed countries; and, in contrast, on the inadequacies of most issues relating to the fight against SCD in Africa.Multiple constraints require an organization based on a network of health professionals working in sickle cell referral centers with specific missions of research, communication, teaching, establishment of guidelines for diagnosis, treatment, and prevention, and the centers of competence that will focus primarily on the screening, diagnosis, and management of SCD patients favoring equity in access to care.

Hydroxyurea (HU) is the only drug which has demonstrated efficacy in terms of reduced incidence of VOC and ACS, reduced need for hospitalization and reduced number of blood transfusion in sickle cell disease. African countries are among those with highest rate of both malaria and sickle cell disease. Despite of that the heterozygous form (HbAS) is known to protect against malaria, there is no documented studies on the clinical effect of HU on malaria. 36 patients aged 4 to 60 years old and treated by HU at the sickle cell disease research and control center of Bamako for at least 2 years. The treated group was compared to a matched untreated sickle cell patients group. During the follow-up, malaria incidence and onset to clinical episode of malaria were compared between treated SCD patients and untreated group. Results of the comparison show that there is no increased incidence of malaria in the treated group after 2 years follow-up (P=0.9). However, a reduction on the time to first malaria episode within the sickle cell population treated with HU was observed (P=0002).Based on this observed reduced time to first malaria episode in HU treated SCD patients, the use of HU in malaria endemic areas needs to be revaluated. It is howether clear that a more comprehensive approach would hopefully reduce the morbidity due to HU use for SCD affected children. Disclosures No relevant conflicts of interest to declare.

The malaria hypothesis proposes a survival advantage for individuals with hemoglobin variants in areas of endemicPlasmodium falciparum malaria. Hemoglobin C (HbC) is a possible example in West Africa, where this hemoglobin has a centric distribution with high frequencies among certain populations including the Dogon ethnic group. To test whether HbC is associated with protection from malaria, we performed a case-control study in the Dogon of Bandiagara, Mali. HbC was present in 68 of 391 (17.4%) of uncomplicated malaria control cases, whereas it was detected in only 3 of 67 cases (4.5%) of severe malaria (odds ratio [OR], 0.22;P = .01). Further, HbC was present in only 1 of 34 cases (2.9%) with cerebral manifestations, the most common presentation of severe malaria in this population (OR, 0.14; P = .03). Episodes of uncomplicated malaria and parasitemias (4800-205 050/μL) were identified in cases of homozygous HbC (HbCC), which indicates thatP falciparum parasites are able to efficiently replicate within HbCC erythrocytes in vivo. These findings suggest that HbC does not protect against infection or uncomplicated malaria but can protect against severe malaria in the Dogon population of Bandiagara, Mali. The data also suggest that the protective effect associated with HbC may be greater than that of HbS in this population.

Complement receptor one (CR1) is a ligand for the rosetting of Plasmodium falciparum infected red cells with uninfected cells. Since CR1 exhibits three known polymorphisms, we studied European-Americans (n = 112) and African-Americans (n = 330) and Malians (n = 158) to determine if genetic differences existed in an area endemic for malaria that could offer a survival advantage. The frequencies of Knops blood group phenotypes McC(b+) and Sl(a-) were greatly increased in Africans vs Europeans. Although the frequency of McC(b+) was similar between Africans from the USA or Mali, the Sl(a-) phenotype was significantly higher in Mali (39% vs 65%, respectively). There was an increased frequency of the largest size (250 kD) of CR1 in Mali, but this did not differ significantly from the USA (P = 0.09). Both cohorts of Africans had higher expression of red cell CR1 than European-Americans but this showed little difference between the USA and Mali groups. Thus, the most important CR1 polymorphism relevant to rosetting of malaria infected cells appears to be the Knops blood group.

Laboratory capacity in the developing world frequently lacks quality management systems (QMS) such as good clinical laboratory practices, proper safety precautions, and adequate facilities; impacting the ability to conduct biomedical research where it is needed most. As the regulatory climate changes globally, higher quality laboratory support is needed to protect study volunteers and to accurately assess biological parameters. The University of Bamako and its partners have undertaken a comprehensive QMS plan to improve quality and productivity using the Clinical and Laboratory Standards Institute standards and guidelines. The clinical laboratory passed the College of American Pathol- ogists inspection in April 2010, and received full accreditation in June 2010. Our efforts to implement high-quality standards have been valuable for evaluating safety and immunogenicity of malaria vaccine candidates in Mali. Other disease-specific research groups in resource-limited settings may benefit by incorporating similar training initiatives, QMS methods, and continual improvement practices to ensure best practices.

Introduction About 80 % of sickle cell disease (SCD) patients live in sub-Saharan Africa whereas most studies on SCD are performed in Europe and America. Hyperhemolysis is thought to play a major role in the pathological process of SCD vasculopathy. Nevertheless, the paradigm of hyperhemolysis is controversial and has never been studied in the African context. We aim to analyze the association between hemolysis and clinical vascular complications among SCD African patients. Methods CADRE is a multinational cohort of SCD African patients aged 3 years and older, included prospectively and explored in a steady state. All patients from the CADRE cohort in Ivory Coast, Cameroun and Mali were included in the present study. Patients were classified in SS-Sβ0 phenotype or SC-Sβ+ phenotype. SCD vascular complications (pulmonary arterial hypertension (PAH), microalbuminuria, leg ulcers, priapism, stroke and osteonecrosis) were assessed using clinical examination, laboratory exams and echocardiography. Hemolysis was measured by a composite score, created with a principal component analysis, that included LDH, hemoglobin and bilirubin rates and clinical icterus. The association between hyperhemolysis (upper quartile of the hemolysis score) and the vascular complications was assessed using multivariate regression analysis in the whole population with further stratification by hemoglobin phenotype. Results We included 2409 patients among which 1751 SS-Sβ0 patients and 658 SC-Sβ+ patients. Compared to SC-Sβ+ patients, SS-Sβ0 patients were younger (15 years old versus 21 years old) and exhibited more leg ulcers (166 (9.5%) versus 24 (3.7%)) and microalbuminuria (573 (42.4%) versus 119 (20.0%)). The hemolysis score was higher in SS-Sβ0 patients as compared to SC-Sβ+ patients (median 1.18 versus 0.43, Figure). After adjustment for age, sex and country, hyperhemolysis was associated with microalbuminuria (OR = 1.59 [1.17-2.16]) and priapism (OR=1.58 [1.01-2.49]) (Table). In SS-Sβ0 patients, hyperhemolysis was associated with microalbuminuria (OR=1.54 [1.10-2.15]) and there was a trend to an association with PAH (OR=1.65 [0.91-2.98]) and priapism (OR=1.50 [0.95-2.39]). In SC-Sβ+ patients, only the association between hyperhemolysis and microalbuminuria remained significant. Sensibility analysis in the adult population showed that hyperhemolysis was significantly associated with all the SCD vascular complications, except for osteonecrosis. Conclusion In African SCD patients, associations between hyperhemolysis and SCD vascular complications were statistically significant but of modest magnitude, and depended on the hemoglobin phenotype. These results suggest that in the African context, hemolysis is not a major determinant of the development of SCD vasculopathy. Table. Associations between SCD complications and hemolysis score (quartiles 1 to 3 versus quartile 4) in the whole population: multivariate analysis with adjustment for age, sex, country and hemoglobin phenotype % of patients with the complication Multivariate analysis N event/N total Quartiles 1 to 3 Quartile 4 OR IC 95% P value PAH* 115/431 6.14 7.08 1.58 0.89-2.83 0.123 Urine albumin/creatinine > 30mg/g 724/1991 24.88 45.36 1.59 1.17-2.16 0.004 Leg ulcer, lifetime 190/2409 6.80 11.01 1.20 0.81-1.78 0.355 Priapism, lifetime ** 160/1095 13.27 17.90 1.58 1.01-2.49 0.046 Stroke, lifetime 25/2409 0.79 1.77 1.32 0.49-3.54 0.578 Osteonecrosis, lifetime 277/2409 11.22 12.28 1.00 0.70-1.43 1.000 PAH: Pulmonary Arterial Hypertension. * Patients from Mali and Cameroun only ** Males only Figure 1. Distribution of the hemolysis score: global population, SS-Sβ0 patients and SC-Sβ+ patients Figure 1. Distribution of the hemolysis score: global population, SS-Sβ0 patients and SC-Sβ+ patients Disclosures No relevant conflicts of interest to declare.

A double blind, randomized and controlled Phase 1 clinical trial was conducted to assess the safety and immunogenicity in malaria-exposed adults of the Plasmodium falciparum blood stage vaccine candidate Apical Membrane Antigen 1-Combination 1 (AMA1-C1)/Alhydrogel with and without the novel adjuvant CPG 7909. Participants were healthy adults 18-45 years old living in the village of Donéguébougou, Mali. A total of 24 participants received 2 doses one month apart of either 80 microg AMA1-C1/Alhydrogel or 80 microg AMA1-C1/Alhydrogel + 564 microg CPG 7909. The study started in October 2007 and completed follow up in May 2008. Both vaccines were well tolerated, with only mild local adverse events and no systemic adverse events judged related to vaccination. The difference in antibody responses were over 2-fold higher in the group receiving CPG 7909 for all time points after second vaccination and the differences are statistically significant (all p0.05). This is the first use of the novel adjuvant CPG 7909 in a malaria-exposed population.

La drepanocytose est l’affection genetique la plus repandue dans le monde plus particulierement en Afrique subsaharienne, consideree a juste titre comme l’une des regions originaires de la mutation drepanocytaire avec de fortes prevalences. Elle est reconnue comme un probleme de sante majeur par la communaute internationale en raison de sa morbidite et de sa mortalite elevees.

Sickle cell disease (SCD) is a multisystem disorder characterized by chronic hemolytic anemia, vaso-occlusive crises, and marked variability in disease severity. Patients require transfusions to manage disease complications, with complements, directed by complement regulatory genes (CR1) and its polymorphisms, implicated in the development of alloantibodies. We hypothesize that CR1 polymorphisms affect complement regulation and function, leading to adverse outcome in SCD. To this end, we determined the genomic diversity of complement regulatory genes by examining single nucleotide polymorphisms associated with Knops blood group antigens. Genomic DNA samples from 130 SCD cases and 356 control Africans, 331 SCD cases and 497 control African Americans, and 254 Caucasians were obtained and analyzed, utilizing a PCR–-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay. Analyzing for ethnic diversity, we found significant differences in the genotypic and allelic frequencies of Sl1/Sl2 (rs17047661) and McCa/b (rs17047660) polymorphisms between Africans, African Americans, and Caucasians ( P < 0.05). The homozygote mutant variants had significantly higher frequencies in Africans and African Americans but were insignificant in Caucasians (80.2% and 59.6% vs 5.9% for Sl1/2; and 36% and 24% vs 1.8% for McCa/b). With SCD, we did not detect any difference among cases and controls either in Africa or in the United States. However, we found significant difference in genotypic ( P < 0.0001) and allelic frequencies ( P < 0.0001) of Sl1/Sl2 (rs17047661) and McCa/b (rs17047660) polymorphisms between SCD groups from Africa and the United States. There was no difference in haplotype frequencies of these polymorphisms among or between groups. The higher frequency of CR1 homozygote mutant variants in Africa but not United States indicates a potential pathogenic role, possibly associated with complicated disease pathophysiology in the former and potentially protective in the latter. The difference between sickle cell groups suggests potential genetic drift or founder effect imposed on the disease in the United States, but not in Africa, and a possible confirmation of the ancestral susceptibility hypothesis. The lower haplotype frequencies among sickle cell and control populations in the United States may be due to the admixture and the dilution of African genetic ancestry in the African American population.

Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.

Severe anemia is one of the major complications of malaria in Africa. We studied 2 populations, one in a village and the second in a periurban area in Mali, to understand the preventable factors in the disease. The 2 correlates of disease were parasitemia above 100 000 parasitized red blood cells per microliter (0.1 × 1012/L) and a low baseline hemoglobin level. All cases of moderate to severe anemia occurred in children under 3.2 years of age. Raising the baseline hemoglobin level and lowering peak parasitemia in infants and young children may reduce the incidence of severe anemia resulting from malarial infection. (Blood. 2004;104: 1198-1200)